Cationic phospholipids for transfection

ABSTRACT

Cationic phospholipids and their use in treating pathogen-associated disease are disclosed. The class of phospholipids comprises the phosphotriester derivatives of phosphoglycerides and sphingolipids. Liposomes comprising one or more of the cationic phospholipids are effective in the lipofection of nuclidic acids and are therefore useful in methods of treating disease.

This is a divisional of application Ser. No. 08/220,376 filed Mar. 29,1994, now U.S. Pat. No. 5,651,981.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel cationic phospholipids and methods formaking them. This invention also relates to novel liposomes andaggregates comprising the phospholipids of the present invention thatare useful for the delivery of nucleic acids and drugs to cells, both invitro and in vivo. This invention also relates to the treatment ofdiseases by gene therapeutics involving transfection with DNA andintroduction into cells of antisense nucleotides, as well as stabletransfection with DNA engineered to become incorporated into the genomeof living cells.

2. Description of the Related Art

The introduction of foreign nucleic acids and other molecules is avaluable method for manipulating cells and has great potential both inmolecular biology and in clinical medicine. Many methods have been usedfor insertion of endogenous nucleic acids into eukaryotic cells. E.g.,Graham and Van der Eb, Virology 52, 456 (1973) (co-precipitation of DNAwith calcium phosphate); Kawai and Nishizawa, Mol. Cell. Biol. 4, 1172(1984) (polycation and DMSO); Neumann et al., EMBO Journal 1, 841 (1982)(electroporation); Graessmann and Graessmann in Microinjection andOrganelle Transplantation Techniques, pp. 3-13 (Cells et al., Eds.,Academic Press 1986) (microinjection); Cudd and Nicolau in LiposomeTechnology, pp. 207-221 (G. Gregoriadis, Ed., CRC Press 1984)(liposomes); Cepko et al., Cell 37, 1053 (1984) (retroviruses); andSchaffner, Proc. Natl. Acad. Sci. USA 77, 2163 (1980) (protoplastfusion). Both transient and stable transfection of genes has beendemonstrated.

Some of the first work on liposome delivery of endogenous materials tocells occurred some twenty years ago. Foreign nucleic acids wereintroduced into cells (Magee et al., Biochim. Biophys. Acta 451, 610-618(1976), Straub et al., Infect. Immun. 10, 783-792 (1974)), as wereforeign lipids (Martin and MacDonald, J. Cell Biol. 70, 515-526 (1976)),Proteins (Magee et al., J. Cell. Biol. 63, 492 (1974), Steger andDesnick, Biochim. Biophys. Acta 464, 530 (1977)), fluorescent dyes(Leventis and Silvius), and drugs (Juliano and Stamp, Biochem. Pharm.27, 21-27 (1978), Mayhew et al., Cancer Res. 36, 4406 (1976), Kimelberg,Biochim. Biophys. Acta 448, 531 (1976)), all using positively chargedlipids.

Of the many methods used to facilitate entry of DNA into eukaryoticcells, cationic liposomes are among the most efficacious and have foundextensive use as DNA carriers in transfection experiments. See,generally, Thierry et al. in Gene Regulation: Biology of Antisense RNAand DNA, p. 147 (Erickson and Izant, Eds., Raven Press, New York, 1992);Hug and Sleight, Biochim. Biophys. Acta 1097, 1 (1991); and Nicolau andCudd, Crit. Rev. Ther. Drug Carr. Sys. 6, 239 (1989) The process oftransfection using liposomes is called lipofection. Senior et al.,Biochim. Biophys. Acta 1070, 173 (1991), suggested that incorporation ofcationic lipids in liposomes is advantageous because it increases theamount of negatively charged molecules that can be associated with theliposome. In their study of the interaction between positively chargedliposomes and blood, they concluded that harmful side-effects associatedwith macroscopic liposome-plasma aggregation can be avoided in humans bylimiting the dosage.

Felgner et al., Proc. Natl. Acad. Sci. USA 84, 7413 (1987), demonstratedthat liposomes of dioleoylphosphatidylethanolamine (DOPE) and thesynthetic cationic lipid N1-(2,3-dioleyloxy)propyl!-N,N,N-trimethylammonium chloride (DOTMA) arecapable of both transiently and stably transfecting DNA. Rose et al.,BioTechiques 10, 520 (1991), tested lipofection with liposomesconsisting of DOPE and one of the cationic lipidscetyldimethylethylammonium bromide (CDAB), cetyltrimethylethylammoniumbromide (CTAB), dimethyldioctadecylammonium bromide (DDAB),methylbenzethonium chloride (MBC) and stearylamine. All of the liposomes(except that with CTAB) successfully transfected DNA into HeLa cells. Athigh concentrations, however, CDAB and MBC caused cell lysis. Only DDABwas found to be effective in mediating efficient DNA transfection into avariety of other cell lines. Malone et al., Proc. Natl. Acad. Sci. USA86, 6077 (1989), successfully transfected RNA, in vitro, into a widevariety of cells lines. Zhou and Haung, J. Controlled Release 19, 269(1992), disclosed successful lipofection by DOPE liposomes stabilized inthe lamellar phase by cationic quaternary ammonium detergents. Theauthors noted, however, that the relatively high cytotoxicity of thesecompounds would limit their use in vivo.

Hawley-Nelson et al., Focus 15, 73 (1990, BRL publications), disclosedthe cationic lipid "LIPOFECTAMINE", a reagent containing2,3-dioleyloxy-N-2(sperminecarboxy-amido)ethyl!-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA). "LIPOFECTAMINE" was found to have highertransfection activity than several monocationic lipid compounds("LIPOFECTIN", "LIPOFECTACE", and DOTAP) in six of eight cell typestested. They observed toxicity when both lipid and DNA were included inthe same mixture.

Both Farhood et al., Biochim. Biophys. Acta 1111, 239 (1992), and Gaoand Huang, Biochem. Biophys. Res. Comm. 179, 280 (1991), disclosecationic derivatives of cholesterol as components of liposomes capableof transfecting cells in vitro.

Liposomes comprising cationic lipids may also find use as carriers forgene therapy in in vivo applications. Some of the first in vivoapplications of delivery of endogenous materials via liposomes wasdemonstrated twenty years ago. See, e.g., Straub et al., supra, andMagee et al., supra, (nucleic acids), and Mayhew et al., supra (drugs),

Holt et al., Neuron 4, 203 (1990), describe a DOTMAdi-oleoxylphosphatidylethanolamine liposome that successfullytransfected a vector expressing luciferase cDNA into embryonic brain ofXenopus in vivo.

Malone, Focus 11, 4 (1989, BRL publications), reported a similar studyon Xenopus neural tissue as did Ono et al., Neurosci. Lett. 117, 259(1990), in mouse brain.

Brigham et al., Am J. Med. Sci. 298, 278 (1989), disclosed intravenousinjection of "LIPOFECTIN" and chloramphenicol acetyl transferase (CAT)plasmid into mouse lungs.

Nabel et al., Science 249, 1285 (1990), reported the expression of aβ-galactosidase gene in a specific arterial segment in vivo in Yucatanpigs by DNA transfection with cationic liposomes. Lim et al.,Circulation 83, 2007 (1991), disclosed in vivo gene transfer of reportergenes (β-galactosidase and luciferase) into arteries of dogs usingcationic liposomes.

Hazinski, Sem. Perinatol. 16, 200 (1992) disclosed cationicliposome-mediated transfer of fusion reporter genes to the epithelialcells and transient protein expression via direct injection ofDNA-liposome solution into the trachea.

Yosimura et al., Nucleic Acids Res. 20, 3233 (1992) demonstratedsuccessful in vivo lipofection of the cystic fibrosis trans-membraneconductance regulator gene (CFTR) into airway epithelium of mice usingthe cationic liposome "LIPOFECTIN". Hyde et al., Nature 362, 250 (1993),also disclosed lipofection of CFTR using "LIPOFECTIN". They demonstratedsuccessful delivery of the gene to epithelia of the airway and toalveoli deep in the lung of transgenic mice.

Several cationic amphiphiles have been reported as transfection agents.Ballas et al., Biochim. et Biophys. Acta 939, 8 (1988), reported thesuccessful lipofection of tobacco mosaic virus RNA into tobacco andpetunia protoplasts via liposomes composed of phosphatidylcholine (PC),cholesterol, and the hydroxyl form of the quaternary ammonium detergentdiisobutylcresoxyethoxyethyldimethylbenzylammonium (DEBDA OH!).Liposomes lacking the quaternary ammonium detergent practically failedto transfect the RNA. Importantly, Ballas et al. also observed that RNAand DNA complexed to liposomes bearing DEBDA OH--! were highly resistantto added RNAses and DNAses.

Pinnaduwage et al., Biochim. et Biophys. Acta 985, 33 (1989), disclosedthe lipofection of pSV2 CAT plasmid DNA into mouse L929 fibroblastsusing sonicated liposomes comprising DOPE and a quaternary ammoniumdetergent (dodecyl-, tetradecyl-, or cetyl-trimethylammonium bromide).Pinnaduwage et al. note, however, that a major drawback of using singlechain amphiphiles such as detergents for drug delivery is theirtoxicity.

Taylor et al., Nucleic Acids Res. 20, 4559-4565 (1992) successfullytransfected both RNA ribozymes and chimeric RNA-DNA ribozymes with"LIPOFECTIN".

Leventis and Sivius, Biochim. et Biophys. Acta 1023, 124 (1990),disclosed several cationic amphiphiles based on a hydrophobiccholesteryl or dioleoylglyceryl moiety whose hydrophobic and cationicportions are linked by ester bonds, which should facilitate degradationin animal cells. Leventis and Sivius demonstrated successful lipofectionof plasmid pSV2 CAT into CV-1 and 3T3 cells using liposomes containingthe cationic amphiphiles1,2-dioleoyl-3-(4'-trimethylammonio)butanoyl-sn-glycerol (DOTB), DOTAPand cholesteryl (4'-trimethylammonio)butanoate (ChoTB).

Duizguines and Felgner, Methods in Enzymology 221, 303 (1993), describemethods for transfection of nucleic acids. They teach that whenpreparing complexes of DNA and "LIPOFECTIN" for transfection, a netpositive charge is desired, and the corresponding ratio of the weight oflipid to nucleic acid is about 4-10. They warn, however, thatoptimization should be undertaken for each cell line to be transformed.

The precise way in which nucleic acids and phospholipids (and otheramphiphiles) interact and the structure formed before and during thetransfection process is not well understood. Commonly, the nucleic acidsare said to be entrapped within a lipid bilayer, which is the classicdefinition of "liposome." There is also a belief, however, that thenucleic acid does not become entrapped, but forms some other sort ofaggregate with the phospholipids. See, e.g., Smith et al., Biochim.Biophys. Acta 1154, 327 (1993), for several models of lipid/nucleic acidinteraction. Maccarrone et al., Biochem. Biophys. Res. Comm. 186, 1417(1992), disclosed that liposome-DNA aggregate size and shape was afunction of the ratio of the amount DNA to that of phospholipid. Theyconcluded that DNA binds to the outer surface of liposomes, which thencluster into irregular spherical aggregates. They also noted thatplasmid length had no effect on binding to liposomes. Gershon et al.,Biochem. 32, 7143-7151 (1993) examined the fluorescence of ethidiumbromide in the presence of DOTMA and DNA. They observed an abrupt dropin its fluorescence when DNA/ethidium bromide is titrated with DOTMA tothe point of near electrical neutrality. Electron microscopy of thecomplex revealed an abrupt condensation of the DNA at the point ofneutrality. It is evident from these experiments that, at least forDOTMA, but probably for most cationic lipids as well, that the structureof the complex changes at charge neutrality, and concomitantly the DNAbecomes very compactly organized into a structure that is evidentlyquite different from a vesicular liposome. Legendre and Szoka, Pharm.Res. 9, 1235 (1992), studied in vitro lipofection using a DOTMA:DOPEliposome and concluded that the liposome probably uses at least twopathways to introduce DNA into cells: fusion with the plasma membraneand endocytosis.

It should be recognized that virtually all of the compounds describedthus far in the literature as "cationic lipids" are, in fact, cationicamphiphiles or cationic detergents. The term "lipid" refers to a naturalproduct. Most lipids contain fatty acids as their major hydrophobiccomponent, although some (such as cholesterol, sphingolipids andpolyisoprenoids) have other hydrophobic structures.

Reagent mixtures of phosphatidylethanolamine (PE) with either DOTMA ordioctadecyldimethylammonium bromide (DDMB) are commercially available(e.g., from Promega). DOTMA-based transfection reagents are expensivedue to the synthetic complexity of the cationic lipid, while the simpledetergents are cheap but require dilution of the cationic species withrelatively expensive PE. Both show significant cytotoxicity (especiallysingle-chain compounds). The underlying causes of the cytotoxicity areunclear, but the difficulty or impossibility of metabolizing thesematerials can only exacerbate this problem during long-term use.Cytotoxicity is not a pressing problem for transient transfectionprocedures, but it must be solved prior to the use of liposometransfection in therapeutic applications. Consequently, cheaper, safer,and more effective lipids useful in lipofection technology aredesirable.

As is further described below, the present invention provides novelcompounds having these attributes. This new class of compounds comprisephosphoglyceride derivatives having a modified phosphodiester linkage,wherein a non-bridging oxygen is alkylated, producing a phosphatetriester. In so alkylating the phosphate moiety, the negative charge onthe oxygen is eliminated.

Methylation of phosphodiester linkages to produce P(O)-methylderivatives has been reported. Renkonen, Biochim. et Biophys. Acta 152,114 (1968). Although treatment of phosphatidylcholine (PC) withdiazomethane yields dimethyl phosphatidic acid with loss of the cholineresidue, O-methyl phosphatidylcholinium has been isolated in low yieldfrom this reaction in the presence of triethylammonium hydrochloride asproton donor. Diazomethane on a preparative scale is extremelyhazardous, however, and the number of readily available diazoalkanes islimited. We also note that methyl phosphatidylcholinium is relativelyunstable. Thus, a more efficient method for producing phosphate triesterderivatives of phosphatidylcholine is desirable.

SUMMARY OF THE INVENTION

We disclose here a novel class of cationic phospholipids that arecapable of generating liposomes. The phospholipids of the presentinvention are derived from parent phosphoglyceride compounds having thestructures: ##STR1## wherein one or both of R¹ and R² typically arehydrocarbon chains having from 1 to about 24 carbons, optionallysubstituted with a dansyl, NBD (nitrobenzofurazan), DPH(1,6-diphenyl-1,3,5-hexatriene), carbocyclic group or a heterocyclicmoiety, s and t are independently 0 or 1, R³ is hydrogen or methyl, andn is 0, 1, 2, or 3. In a preferred embodiment of the present invention,the parent phosphoglyceride compound is naturally occurring. In aparticularly preferred embodiment, the compound is phosphatidylcholine.

The novel phospholipids of the present invention obtained by alkylatingthe above reactant compounds have the following structure: ##STR2##wherein all definitions described above for the parent compound hold, R⁴is an optionally substituted C₁ to about C₂₄ hydrocarbon chain.

These lipids are attractive for a number of reasons. First, we havefound that liposomes and aggregates comprising these cationicphospholipids are effective in the transfection of nucleic acids, havingefficacy comparable to that of the commonly used commercial product"LIPOFECTIN". Second, most of the phospholipids of the present inventionare easily made from cheap and readily available starting materials in aone step synthesis. And finally, most of the phospholipids of thepresent invention are derived from a naturally occurring parentcompound, thereby providing a means for cellular metabolism andminimizing the cytotoxicity of these compounds under chronicadministration.

We also disclose compounds generated from sphingophospholipids,phospholipids having a phosphorylcholine polar group, identical to thatof phosphatidylcholine, but with ceramide in place of dialkylglycerol inthe hydrophobic portion of the molecule. Sphingophospholipids arealkylated in the same way as the phosphoglycerides discussed above,generating the corresponding cholinium compounds. The general structureof the sphingophospholipids is: ##STR3## where R₅ is a fatty acid orrelated carboxylic acid of the same type as R¹, supra. Alkylation is atthe same position (on the phosphate oxygen) as in the phosphoglycerides.

We also present a novel method of synthesizing the cationicphospholipids of the present invention. The preferred method comprisesalkylating a parent phospholipid compound (having a structure definedabove) by contacting it with a R³ -trifluoromethanesulfonate (triflate)in a suitable solvent, wherein R⁴ is defined above. In a preferredembodiment, the solvent is ether. This method is both cheap and easy,due, in part, to the wide availability and low cost of many of theparent phospholipid compounds (e.g., sphingophospholipids andphosphoglycerides), easy synthesis of alkyl triflates, and the rapidreaction that occurs at or near room temperature. In a preferredembodiment of the present invention, the parent phospholipid compound isphosphatidylcholine, which may be obtained from egg yolk, other naturalsources, or synthesized.

Coupled with the general availability of the parent phospholipid, thismethod for synthesis provides a broad spectrum of phosphoniumderivatives having polar head groups and lipid side chains varying inboth their steric and electronic properties, particularly hydrophobicityand hydrophilicity. Thus, the methods and compounds of the presentinvention essentially allow one to tune the properties of a phospholipidto nearly any desired state.

The present invention also provides for improved methods of transfectionusing the phospholipids of the present invention. Using standardtechniques, nucleic acids can be transfected into cells, in vitro or invivo, with high efficiency. The present invention further provides forimproved methods of drug delivery.

The present invention also provides for improved methods of treatingpatients having diseases or ailments amenable to treatment with nucleicacids, oligonucleotides or drugs. Such diseases include those arisingfrom infection with a pathogen, in which case treatment may compriseadministration of an antisense oligonucleotide targeted to an endogenousnucleic acid within the pathogen that is essential to a developmental,metabolic, or reproductive function of the pathogen or by delivery of adrug. Other diseases include those arising from a DNA deficiency. Suchdeficiency may be the lack of essential DNA or a mutation (such as adeletion, alteration, or addition of one or more nucleotides) resultingin under- or over-expression of a gene or expression of an abnormalprotein. In such a case, treatment may comprise lipofection of normalnucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the synthetic method for alkylating phosphoglycerides toproduce O-- substituted derivatives.

FIG. 2 is an autoradiogram showing the results of a chloramphenicolacetyltransferase assay of extracts of L cells and Rcho-1 cellstransfected with RSVCAT DNA.

FIG. 3 presents the results of a CAT assay of the relative transfectionefficiency of NBD-EtPC⁺.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel cationic phospholipids, liposomescomprising these phospholipids, liposome nucleic acid aggregates,methods for transfecting nucleic acids in vitro and in vivo comprisingcontacting a cell or cells with the liposome-nucleic acid aggregates,and methods for treating diseases arising from infection or a DNAdeficiency treatable by in vivo delivery of nucleic acids.

In the first aspect of the present invention, novel cationicphospholipids are disclosed. These cationic phospholipids are capable offorming liposomes and transfecting nucleic acids into cells. Anattractive feature of this class of compounds is that they can besynthesized from cheap, readily obtainable parent phosphoglyceridecompounds. These phosphoglyceride compounds have the followingstructure: ##STR4## wherein R¹ and R² independently are H or C₁ to aboutC₂₄ straight or branched alkyl, alkenyl, or alkynyl chains optionallysubstituted with a dansyl, NBD, DPH, carbocyiclic aromatic, orheterocyclic moiety,

R³ is hydrogen or methyl,

n is 0, 1, 2, or 3,

s is 0 or 1, and

t is 0 or 1,

provided that R¹ and R² are not both H and that when R¹ is H, s is 0,and that when R² is H, t is 0.

In a preferred embodiment of the present invention, the parentphosphoglyceride compound is naturally occurring or readily derived froma naturally occurring compound. In a particularly preferred embodiment,the parent phosphoglyceride compound is phosphatidylcholine.Phosphatidylcholine is extremely cheap and easily obtainable from eggyolk.

The novel phospholipids of the present invention obtained from the abovereactant compounds have the following structure: ##STR5## wherein alldefinitions described above for the parent compound hold, R⁴ is C₁ toabout C₂₄ straight or branched alkyl, alkenyl, or alkynyl chainsoptionally substituted with dansyl, NBD, DPH, carbocylclic aromatic, orheterocyclic moiety, or R⁴ is a C₁ to about C₆ straight or branchedchain ester, aldehyde, ketone, ether, haloalkyl, azidoalkyl, ortetraalkylammonium.

As used herein, the term "alkenyl" refers to a moiety having from 1 to 6double bonds. "Alkynyl" refers to a moiety having 1 or 2 triple bonds. A"branched chain" has from 1 to 4 branching methyls and up to 2cyclopropyl groups. A "carbocyclic aromatic" has up to 4 rings, whichmay be fused or spiro, and 5 or 6 carbon atoms per ring. "Heterocyclicmoiety" means 1 to 3 rings having 5 or 6 atoms in the ring or rings andup to 2 oxygen or nitrogens per ring.

In a preferred embodiment, the compound is the phosphotriesterderivative of a naturally occurring compound. In a particularlypreferred embodiment, the compound is the phosphotriester derivative ofphosphatidylcholine. In another preferred embodiment, the compound isthe ethyl phosphotriester derivative of phosphatidylcholine. In yetanother preferred embodiment, R¹ or R² is an NBD substituted C₆ -C₁₂alkyl. As is described more fully below, we have prepared a number ofthese compounds.

A second group of novel cationic phospholipids is derived from a classof sphingophospholipid related compounds having the general structure:##STR6## where R⁵ is a fatty acid or related carboxylic acid having thesame structure as R¹, supra. The sphingophospholipids are alkylated inthe same manner as the phosphoglyceride compounds. The alkyl group addsto the same position on the phosphate oxygen as on the phosphoglyceridesto yield: ##STR7##

As will be described more fully below, it has been found that thesecationic phospholipids of the present invention are effective agents fortransfecting nucleic acids and drugs.

In a second aspect of the present invention, a novel method ofsynthesizing the aforementioned phospholipids is disclosed. Thesynthetic method of the present invention comprises mixing an R⁴substituted trifluoromethanesulfonate (triflate) with a parentphosphoglyceride or sphingophospholipid in a suitable solvent, whereinR⁴ is C₁ to about C₂₄ straight or branched alkyl, alkenyl, or alkynylchains optionally substituted with a dansyl, NBD, DPH, carbocylclicaromatic, or heterocyclic moiety, or R⁴ is a C₁ to about C₆ straight orbranched chain ester, aldehyde, ketone, ether, haloalkyl, azidoalkyl, ortetraalkylammonium. By suitable solvent it is meant any solvent capableof dissolving both reactant species and that itself does not undergo achemical reaction with the starting materials, intermediates orproducts. Suitable solvents will be recognized or can be determined in aroutine manner by those skilled in the art. In a preferred embodiment,the solvent is diethyl ether. The reaction proceeds rapidly at roomtemperature to yield nearly instantaneously the phospholipids describedabove. The products can then be purified by any suitable means. Apreferred method of purification is by simple batch chromatography onSiO₂.

Under neutral conditions, the reaction proceeds by transferring thetriflate substituent to a non-bridging oxygen of the phosphate to form aphosphotriester. In the presence of a sterically hindered base (e.g.,collidine) and when R³ is hydrogen, both the phosphate oxygen and theethanolamine nitrogen will be substituted. See FIG. 1.

A preferred method of the present invention comprises reaction of anethereal solution of phosphoglyceride parent compound with analkyl-trifluoromethanesulfonate. The result is a nearly instantaneous,clean conversion to the substituted phosphotriester derivative of thephosphoglyceride parent compound as the triflate salt. Purification isthen by simple batch chromatography on SiO₂, which affords purealkylphospholipid triester in 85% isolated yield on a 1-gram scale. In apreferred embodiment, the alkyl group is an ethyl moiety.

Substituted triflates are easily made according to the followingreaction ether ##STR8## Triflic anhydride is available from Alrich®, forexample.

Using the methods of the present invention, we have conducted thefollowing syntheses: ##STR9##

    ______________________________________    Cmpd                        Cmpd    Product    #      Reactant             #       (R.sup.4)    ______________________________________    1      R.sup.1R.sup.2C.sub.17 H.sub.33                                2       CH.sub.3                                3       CH.sub.2 CH.sub.3                                10      (CH.sub.2).sub.6 Br                                11      (CH.sub.2).sub.6 N.sub.3    4      R.sup.1R.sup.2C.sub.11 H.sub.23                                7       CH.sub.2 CH.sub.3    5      R.sub.1C.sub.17 H.sub.33                                8       CH.sub.2 CH.sub.3           R.sub.2CH.sub.3    6      R.sub.1C.sub.17 H.sub.33                                9       CH.sub.2 CH.sub.3    ______________________________________     wherein t=s= 1, n=3, and R.sup.3 =CH.sub.3. Compounds 10 and 11 were     prepared at -78° C. The functional groups in compounds 10 and 11     can be modified to allow for conjugation of the cationic lipid with other     molecules.

The availability of both naturally occurring and synthetic phospholipidscoupled with the wide availability of alkyl triflates provides a unifiedroute to the synthesis of a diverse array of cationic lipids. Thisshould provide the ability to tune the molecular and biologicalproperties of the transfection reagent and provide a means ofinvestigating the molecular basis of lipid-based transfection. Finally,the basis of the cationic lipid in a natural phospholipid skeletonprovides for the attenuation of long-term cytotoxicity through metabolictransformation of the compound to a natural product.

While the preferred method of producing the compounds of the presentinvention is by the novel triflate method described, those of ordinaryskill in the art will recognize that other art recognized methods ofsynthesis may be used. For instance, other alkylating agents that may beused include alkyl iodides, alkyl tosylates, dialkylsulfates, andtrialkyl oxonium wherein the anion is BF₄ ⁻.

Because of their unique steric and electronic properties, the modifiedcationic phospholipids synthesized by the foregoing methods generate anovel class of liposomes that are very effective in the lipofection ofnucleic acids. As used herein, the term "liposome" is meant to encompassall compositions of phospholipids (and/or other amphiphiles) thataggregate in aqueous solution, including micelles, as well as cubic andhexagonal phases.

The liposomes of the present invention comprise one or more of thephospholipids of the present invention. Liposomes according to theinvention optionally have one or more other amphiphiles. The exactcomposition of the liposomes will be depend on the particularcircumstances for which they are to be used. Those of ordinary skill inthe art will find it a routine matter to determine a suitablecomposition. The liposomes of the present invention comprise at leastone phospholipid of the present invention. In a preferred embodiment,the liposomes of the present invention consist essentially of a singletype of phospholipid according to the invention. In another preferredembodiment, the liposomes comprise mixtures of compounds according tothe present invention. In yet another preferred embodiment, theliposomes of the present invention comprise one or more phospholipids ofthe present invention in mixture with one or more natural or syntheticlipids, e.g., cholesterol. In still another preferred embodiment, theliposomes consist essentially of the ethyl phosphotriester derivative ofphosphatidylcholine. It is a routine matter, using techniques well knownin the art, to determine an appropriate and optimal ratio of componentswhere mixtures of phospholipids are used.

Liposomes are constructed by well known techniques. E.g., LiposomeTechnology, Vols. 1-3 (G. Gregoriadis, Ed., CRC Press, 1993) Lipids aretypically dissolved in chloroform and spread in a thin film over thesurface of a tube or flask by rotary evaporation. If liposomes comprisedof a mixture of lipids is desired, the individual components are mixedin the original chloroform solution. After the organic solvent has beeneliminated, a phase consisting of water optionally containing bufferand/or electrolyte is added and the vessel agitated to suspend thelipid. The suspension is then subjected to ultrasound, either in anultrasonic bath or with a probe sonicator, until the particles arereduced in size and the suspension is of the desired clarity. Fortransfection, the aqueous phase is typically distilled water and thesuspension is sonicated until nearly clear, which requires some minutesdepending upon conditions, kind, and quality of the sonicator. Commonly,lipid concentrations are 1 mg/ml of aqueous phase, but could easily be afactor of ten higher or lower.

In a third aspect of the invention, a novel method of lipofection isprovided comprising contacting cells to be transformed with a solutionof liposome-nucleic acid aggregates. Liposome-nucleic acid aggregatesmay be prepared by adding an appropriate amount of nucleic acid to aliposome solution. For transfection, the weight ratio of cationic lipidto DNA is from slightly over 1:1 to perhaps 10:1. The amount of DNA canvary considerably, but is normally a few to a few tens of micrograms perstandard culture dish of cells. Conditions may vary widely, and it is aroutine matter and standard practice to optimize conditions for eachtype of cell, as suppliers of commercial materials recommend.Optimization involves varying the lipid to DNA ratio as well as thetotal amount of aggregate.

As has been noted, there is currently some uncertainty regarding theprecise way in which nucleic acids and phospholipids (and otheramphiphiles) interact. In addition, the structure formed both before andduring the transfection process is not definitively known either. Thepresent invention, however, is not limited by the particular structuraltype of complex formed by the liposomes of the present invention and thenucleic acids to be transfected. As used herein, therefore, the phrase"liposome-nucleic acid aggregate" means any association of liposomes andnucleic acids that is capable of lipofection.

The lipid-nucleic acid aggregate is added to the cells, in culturemedium, and left for some tens of minutes to several hours to perhapsovernight. Usually serum is omitted from the culture medium during thisphase of transfection. Subsequently, the medium is replaced with normal,serum-containing medium and the cells are incubated for hours to days orpossibly cultured indefinitely.

Both P(O) methylated phosphatidylcholine (MePC⁺, 2) and ethylatedphosphatidylcholine (EtPC⁺, 3) are effective mediators of DNAtransfection into eukaryotic cells, showing efficiency in RSV-CATtransfections (e.g., Gorman et al., Mol. Cell. Biol. 2, 1044 (1982); deWet et al., Mol. Cell. Biol. 7, 725 (1987)) into L cells and Rcho-1cells equivalent to or better than that observed with "LIPOFECTIN"(Example 3 and FIG. 2). Unlike existing transfection reagents made fromcationic detergents, MePC⁺ (2) and EtPC⁺ (3) are effective transfectionreagents in the absence of phosphatidyethanolamine (PE). The reasons forthis difference are not clear at present, but recent studies haveindicated that the PE co-lipid requirement for "LIPOFECTIN" varies withthe cell type and culture age. Jarnagin et al., Nucl. Acids Res. 20,4205 (1992). Interestingly, the fluorescent nitrobenzofurazan (NBD)tagged lipid 9 mediated transfection better than EtPC⁺ (3), as cellstransfected using mixtures of EtPC⁺ (3) and 9 showed increasing CATactivity as the proportion of 9 in the mixture increased. This suggeststhat the nature of the fatty acid substituent in the phosphoglycerideparent compound plays an important role in determining DNA transfectionefficiency. The ability to prepare a diverse array of cationicphosphatidylcholine derivatives using the methods of the presentinvention will allow a more systematic investigation of the structuralfactors involved in DNA transfection by cationic liposomes and providefor a wider variety of cationic liposomes useful for lipofection.

Myriad nucleic acids may be associated with the liposomes of the presentinvention and transfected. These include DNA, RNA, DNA/RNA hybrids (eachof which may be single or double stranded), including oligonucleotidessuch as antisense oligonucleotides, chimeric DNA-RNA polymers, andribozymes, as well as modified versions of these nucleic acids whereinthe modification may be in the base, the sugar moiety, the phosphatelinkage, or in any combination thereof.

From the foregoing it will be clear to those skilled in the art that theliposomes of the present invention are useful for both in vitro and invivo application. The liposomes of the present invention will find usefor nearly any in vitro application requiring transfection of nucleicacids into cells--one such example being in the process of recombinantproduction of a protein.

The nucleic acids may comprise an essential gene or fragment thereof ofwhich the target cell or cells is deficient in some manner, such aslacking the gene or wherein the gene is mutated resulting in under- orover-expression. The associated nucleic acids may also compriseantisense oligonucleotides. Such antisense oligonucleotides may beconstructed to inhibit expression of a target gene. The foregoing arebut examples of nucleic acids that may be used with the presentinvention and are not intended and should not be construed to limit theinvention in any way. Those skilled in the art will appreciate thatother nucleic acids will be suitable for use in the present invention aswell.

The liposomes of the present invention are also useful for drugdelivery. As used herein, the term "liposome-drug aggregate" means anyassociation of liposomes and drugs capable of delivering the drug tocells. The term "drug," as used herein, refers to any non-nucleic acidcompound that is or can be used as a therapeutic, be it protein ornon-protein in nature. The efficiency of delivery of drugs is improvedgreatly if the drug is hydrophobic. The uptake of cationic drugs may beincreased if one includes a counterion such as a fatty acid or if theliposomes are made up in an aqueous solution of high ionic strength. Theparticular liposome and encapsulation process will depend upon the drug.It is a routine matter, however, to determine the appropriate conditionsfor drug delivery using liposomes.

Phospholipid-assisted drug delivery may be accomplished in the followingmanner. For drugs that are soluble in organic solvents, such aschloroform, the drug and cationic lipid are mixed in solvents in whichboth are soluble, and the solvent is then removed under vacuum. Thelipid-drug residue is then dispersed in an appropriate aqueous solvent,which, in a preferred embodiment, is sterile physiological saline. Thesuspension then may optionally be subjected to up to several freeze/thawcycles. It is then sonicated, either merely to reduce the coarseness ofthe dispersion or to reduce the particle size to 20-30 nm diameter,depending upon whether large or small particle size is most efficaciousin the desired application. For some applications, it may be mosteffective to generate extruded liposomes by forcing the suspensionthrough a filter with pores of 100 nm diameter or smaller. For someapplications, inclusion of cholesterol or natural phospholipids in themixture used to generate the lipid-drug aggregate may be appropriate.The liposome-drug aggregate may then be delivered in any suitablemanner.

For drugs that are soluble in aqueous solution and insoluble in organicsolvents, the lipid mixture to be used for the lipid dispersion orliposomes is coated on the inside surface of a flask or tube byevaporating the solvent from a solution of the mixture. In general, forthis method to be successful, the lipid mixture must be capable offorming vesicles having single or multiple lipid bilayer walls andencapsulating an aqueous core. The aqueous phase containing thedissolved drug, preferably a physiological saline solution, is added tothe lipid, agitated to generate a suspension, and then optionally frozenand thawed up to several times. If it is desired to generate smallliposomes, the suspension is subjected to ultrasonic waves for a timenecessary to reduce the liposomes to the desired average size. If largeliposomes are desired, the suspension is merely agitated by hand or on avortex mixer until a uniform dispersion is obtained, i.e., untilvisually observable large particles are absent. If the preparation is tohave the drug contained only within the liposomes, then the drug in theaqueous phase is eliminated by dialysis or by passage through agel-filtration chromatographic column (e.g., agarose) equilibrated withthe aqueous phase containing all normal components except the drug.Again, the lipid mixture used may contain cholesterol or naturalphospholipids in addition to the cationic compounds of the presentinvention. The liposome-drug aggregate may then be delivered in anysuitable manner.

The fourth aspect of the invention comprises novel methods of treatingdiseases arising from infection by a pathogen or from an endogenous DNAdeficiency. These methods comprise administering a liposome-nucleic acidaggregate and/or liposome-drug aggregate solution to a mammal sufferingfrom a pathogenic infection or DNA deficiency. If the disease is theresult of infection by a pathogen, the nucleic acid may be, for example,an antisense oligonucleotide targeted against an DNA sequence in thepathogen that is essential for development, metabolism, or reproductionof the pathogen. If the disease is a DNA deficiency (i.e., whereincertain endogenous DNA is missing or has been mutated), resulting inunder- or over-expression, the nucleic acid may be the normal DNAsequence.

Several methods of in vivo lipofection have been reported. In the caseof whole animals, the lipid-nucleic acid aggregate may be injected intothe blood stream, directly into a tissue, into the peritoneum, instilledinto the trachea, or converted to an aerosol, which the animal breathes.Zhu et al., Science 261, 209-211 (1993) describe a single intravenousinjection of 100 micrograms of a mixture of DNA andDOTMA:dioleoylphosphatidylethanaolamine that efficiently transfectedvirtually all tissues. Nabel et al., supra, used a catheter to implantliposome-DNA aggregates in a blood vessel wall, resulting in successfultransformation of several cell types, including endothelial and vascularsmooth muscle cells.

Stribling et al., Proc. Natl. Acad. Sci. USA 89, 11277-11281 (1992),demonstrated that aerosol delivery of a chloramphenicolacetyltransferase (CAT) expression plasmid complexed to cationicliposomes produced high-level, lung-specific CAT gene expression in micein vivo for at least 21 days. They described the following procedure:Six milligrams of plasmid DNA and 12 μmol of DOTMA/DOPE liposomes wereeach diluted to 8 ml with water and mixed; equal volumes were thenplaced into two Acorn I nebulizers (Marquest, Englewood, Colo.); animalswere loaded into an Intox small-animal exposure chamber (Albuquerque)and an air flow rate of 4 L/min was used to generate the aerosol (about90 min were required to aerosolize this volume) the animals were removedfrom the chamber for 1-2 hours and the procedure was repeated. Thisprotocol is representative of the aerosol delivery method.

The following Examples are presented for illustrative purposes only andare not intended, and should not be construed, to limit the invention inany manner.

EXAMPLES Example 1

Synthesis of O-Methyl and O-Ethyl Phosphatidylcholine

Phosphatidylcholine was obtained as a CHCl₃ solution (Avanti PolarLipids, Inc., Alabaster, Ala.). Methyl trifluoromethanesulfonate andethyl trifluoromethanesulfonate were obtained from Aldrich Chemical Co.(St. Louis, Mo.) and distilled prior to use. Ethyl ether was distilledfrom lithium aluminum hydride prior to use.

The triflate ester (1.0 mmol) was added dropwise to a solution ofphosphatidylcholine (1.0 mmol) in 20 ml of ethyl ether stirred under annitrogen atmosphere. Thin-layer chromatographic analysis (SiO₂, 65:25:4CHCl₃ :CH₃ OH:H₂ O; detection with phosphomolybdate spray) indicatedcomplete conversion of phosphatidylcholine (Rf=0.25) to alkyl PC⁺ (R_(f)=0.60) within 30 minutes. The reaction mixture was poured onto a pad ofSiO₂ (4 cm diameter by 2 cm high) and the ether was pulled through byaspirator vacuum. The SiO₂ pad was washed 3 times with 10 ml portions ofCHCl₃ :CH₃ OH (10:1). Product-containing fractions were pooled andevaporated in vacuo.

The methyl phosphotriester was obtained in 85% yield. The ethylphosphotriester was obtained in 80% isolated yield after a reactionperiod of 4 hours.

Analysis of the methyl phosphotriester of phosphatidylcholine by 600 MHz¹ HNMR revealed a pair of doublets (3H, J=12 Hz) at δ 3.85,characteristic of the expected diastereomeric mixture of phosphatemethyl esters.

Example 2

Liposome Metabolism and Stability

The metabolism of these cationic lipids was investigated using thefluorescent derivative 9. Mouse L-cell cultures were treated with thefluorescent derivative 2, with and without DNA, in the same way as thestandard transfection procedure was carried out in Example 3, infra.After 24 hours of incubation, the cells were scraped from the plate andcentrifuged to isolate them as a wet pellet. Lipid was extracted fromthe cells with milliliter portions of chloroform:methanol (2:1). Thechloroform lower phase of the extract was removed and concentrated. Thinlayer chromatography (TLC) of the concentrated extract on silica gelplates with chloroform:methanol:water (65:25:4) revealed a major spot inthe position of the original fluorescent lipid and a second, lessintense spot with an R_(f) that was approximately that expected for12-NBD-aminodecanoic acid. Based on the relative fluorescent intensityof the two spots, about one third of the original compound washydrolyzed in approximately the time required for the typicaltransfection of cultured cells.

In the absence of cells, aqueous EtPC⁺ (3) is quite stable; using thinlayer chromatography, no measurable degradation is observed afterseveral days at room temperature. Samples have beeen kept in chloroformat -20° C. for more than 2 years without showing signs of degradation.

To further examine the metabolism of these lipids, 9 was treated withpurified phospholipases obtained from various sources following standardprocedures. W. W. Christie, Lipid Analysis (2nd ed., Pergamon Press,Oxford, 1982). Typically, 50-100 micrograms of 9 in 50-250 μl of ethylether were shaken with 50-250 μl of enzyme in buffer containing, ifappropriate for the function of the enzyme, calcium chloride. Severalmicroliter samples were taken at intervals for TLC analysis on silicagel plates with chloroform:methanol:water orchloroform:methanol:concentrated ammonia, both 65:25:4. No detectablereaction was observed with phospholipase C from Clostridium perfringens.Phospholipase D from cabbage, brussels sprouts, peanut, and Streptomyceschromofuscus showed very slow degradation of 2 compared with that of PC.All enzymes except that extracted from brussels sprouts (by us) werecommercial preparations obtained from Sigma (St. Louis, Mo.).

The product of phospholipase D action on 9 has not yet been firmlyidentified, although it is clear that it is neither 1 nor phosphatidicacid. It is possible that the product is phosphatidylethanol based onits chromatographic properties. The choline group is thus not as readilylost from 9 as from phosphatidylcholine under the influence of theenzyme used.

Treatment with phospholipase A2 (Naja naja) produced12-(NBD-amino)dodecanoic acid, although several-fold more slowly thanobserved for the PC control reaction. Cellular metabolism of EtPC⁺ (3)may thus proceed via phospholipase A-mediated hydrolysis, with loss ofthe choline moiety occurring much more slowly via the action ofphospholipase D, although clearly such a conclusion needs to be regardedcautiously because intracellular lipases may exhibit differentactivities than those used in our in vitro assays.

Example 3

Lipofection of Cultured Land Rcho-1 Cells

Mouse L cells were grown in Dulbecco's modified Eagle's medium with 10%calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and glutamine.Rcho-1 cells were grown in RPMI 1640 medium containing 10% heatinactivated fetal calf serum, 50 μM 2-mercaptoethanol, 1 mM sodiumpyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin. Both celllines were grown at 37° C. in a 5% CO₂ atmosphere.

A CH₃ Cl₃ solution of lipid was dried in vacuo and suspended in sterileH₂ O at 10 mg/ml (NBD-tagged lipid was suspended at 1 mg/ml due tolimited solubility) by vortexing. This suspension was sonicated for 30seconds using the microprobe of a Branson 400W sonicator at lowestpower. This suspension was diluted to 1 mg/ml and further treated byextrusion through a 1000 Å ultrafilter to produce more homogeneousliposomes.

An aqueous suspension of liposomes (80 μg of lipid) was mixed with 10 μgof RSV-CAT DNA and kept at ambient temperature for 20 minutes. Cellswere washed with phosphate buffered saline, and the medium was replacedwith either DME or RPMI. Once a cloudy white precipitate had formed inthe liposome-DNA mixture, the solution was added dropwise to the cells.The plates were then gently swirled and incubated 10-24 hrs at 37° C.under 5% CO₂. The medium was replaced by complete medium, and the cellswere harvested 48 hrs after addition of DNA. Transfections involving"LIPOFECTIN" (using 30 μg) were performed according to the publishedprocedures of Gibco-BRL. Cell extracts were prepared and assayed forchloramphenicol acetyltransferase and luciferase activities. Theautoradiogram is presented in FIG. 2. The following table identifies thetransfecting reagents for each lane appearing in the FIG. 2autoradiogram.

    ______________________________________    Cell Type            Lane     Reagents    ______________________________________    Mouse L 1        1000 Å vesicle EtPC.sup.+ (3)            2        1000 Å vesicle EtPC.sup.+ (3) + RSV-CAT DNA            3        sonicated EtPC.sup.+ (3) + RSV-CAT DNA            4        "LIPOFECTIN" + RSV-CAT DNA    Rcho-1  5        1000 Å vesicle EtPC.sup.+ (3) + RSV-CAT DNA            6        sonicated EtPC.sup.+ (3) + RSV-CAT DNA            7        "LIPOFECTIN" + RSV-CAT DNA    ______________________________________

As can be seen from the autoradiogram, EtPC⁺ (3) is as effective atransfecting agent as "LIPOFECTIN".

Following the same protocol, we studied the relative transfectionefficiency of the NBD tagged lipid (9) and that of EtPC⁺ (3). 10 μg ofRSV-CAT and 80 μg of lipid were used. The results are displayed in FIG.3. The following table identifies the contents of the lanes:

    ______________________________________    Lane             Lipid    ______________________________________    1                no lipid    2                NBD-PC    3                EtPC.sup.+    4                1:1 EtPC.sup.+ /NBD-EtPC.sup.+    5                NBD-EtPC.sup.+    ______________________________________

The results presented in FIG. 3 show that NBD-tagged lipid (11) mediatedtransfection somewhat better than EtPC⁺ (3). This suggests that thenature of the fatty acid substituents play an important role indetermining DNA transfection efficiency.

Example 4

Lipofection of Cultured Baby Hamster Kidney and Niemann-Pick (Type A)Fibroblast Cells

EtPC⁺ (3) was used to transfect the protein RAB-7 gene. A vectorcontaining the gene for RAB-7 and the T7 polymerase promoter wasconstructed. It was transfected into baby hamster kidney andNiemann-Pick (type A) cells. The cells were simultaneously infected witha recombinant vaccinia virus containing the T7 polymerase. The ratio ofEtPC⁺ (3) to DNA was 5:1 by weight. Expression of the RAB-7 gene wasdetermined by measuring cells stained with antibodies to the RAB-7protein. Under the conditions used (which were not optimized for EtPC⁺),transfection efficiency (as measured by the percent of cells expressingthe RAB-7 gene) was 30-40% for BHK cells and about 20% for thefibroblasts. These results were very similar to those obtained with thecommercial product "LIPOFECTIN".

Example 5

Lipofection of Cultured Human Erythroleukemia (K562) Cells

The lipofection of cultured human erythroleukemia (K562) cells usingEtPC⁺ (3) was compared to "LIPOFECTIN". Either 10 or 50 μg of EtPC⁺ (3)or 30 μg of "LIPOFECTIN" were mixed with either 5 μg of pRSV-CAT or 5 μgeach of pGAL4-HSF1 and pGAL4-CAT. The lipid-DNA aggregate was then addedto the cells. After 48 hours, the cells were assayed for CAT activityusing the conventional chromatographic radioassay. By visual examinationof the autoradiogram, the results for EtPC⁺ (3) and for "LIPOFECTIN"transductions were very similar.

Example 6

Lipofection of Whole Animals

To lipofect whole animals, the general procedure of Zhu et al., supra,may be used. Per 20 g animal weight, 100 μg of DNA as the appropriatevector, complexed with approximately 600 μg of cationic lipid, isinjected intravenously. It is expected that, for optimal results, theamount of DNA and the ratio of cationic lipid to DNA will need to bevaried and expression of the desired trait evaluated for each amount andratio. Lipid is dispersed in the same way as for in vitro transfection.

For localizing the dose and preferentially treating specific tissues ororgans, various procedures may be applied, such as injections directlyinto the organ or tissue, delivery by canula or injection to ducts,vessels or passages leading to the organ or tissue of interest, ordirect surface application, either manually or through an aerosol vapor,for example, to the lungs.

We claim:
 1. A liposome-nucleic acid aggregate comprising one or morenucleic acids and one or more liposomes, wherein at least one nucleicacid is an antisense oligonucleotide and wherein at least one liposomecomprises one or more cationic phospholipids having the structure:##STR10## wherein R⁴ is a C₁ to C₂₄ straight or branched alkyl, alkenyl,or alkynyl chain optionally substituted with a dansyl, NDB, DPH,carbocylclic aromatic, or heterocyclic moiety, or R⁴ is a C₁ to about C₆straight or branched chain ester, aldehyde, ketone, ether, haloalkyl,azidoalkyl, or tetraalkylammonium,R⁵ is H or a C₁ to about C₂₄ straightor branched alkyl, alkenyl, or alkynyl chain optionally substituted withdansyl, NDB, DPH, carbocylclic aromatic, or heterocyclic moiety.